In Vitro Calcium Imaging in Short-Term and Long-Term Rat Hippocampal Cultures

Take a short-term rat hippocampal neuronal culture representing young neurons and a long-term culture representing aged neurons.

Aged neurons exhibit a higher density of L-type voltage-gated calcium channels, or L-VGCCs, which remain open for a longer duration upon membrane depolarization.

Incubate with a fluorescent calcium indicator that diffuses into the cells, then wash to remove any excess dye.

Place the cultures in a perfusion chamber under a fluorescence microscope.

Introduce a solution containing neurotransmitters that bind to receptors on the neurons, inducing the influx of sodium ions and triggering membrane depolarization.

The depolarization activates the L-VGCCs, enabling calcium influx into the cytoplasm.

In the long-term culture, the higher density and prolonged opening of L-VGCCs leads to calcium overloading, a hallmark of neuronal aging.

Calcium binding to the indicator generates fluorescence, with aged neurons exhibiting higher fluorescence intensity, reflecting increased intracellular calcium levels upon stimulation.